Human trophoblast cells express the immunomodulator progesterone-induced blocking factor.

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1-18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.

Dysregulation of placental endothelial lipase and lipoprotein lipase in intrauterine growth-restricted pregnancies.

CONTEXT: Fetal supply of maternally derived fatty acids requires lipase-mediated hydrolysis of lipoprotein-borne triglycerides and phospholipids at the placental surface. OBJECTIVE: The objective of the study was to test the hypothesis that members of the triglyceride lipase gene (TLG) family are expressed in the human placenta at the maternoplacental (syncytiotrophoblast) and fetoplacental (endothelial cells) interface and that their expression is altered in pregnancy pathologies. DESIGN AND SETTING: Expression of TLG family members in primary placental cells (trophoblast and endothelial cells) and tissues of first-trimester and term human placenta was analyzed by microarrays, RT-PCR, Western blotting, and immunohistochemistry. Their expression was compared between normal pregnancies and those complicated with intrauterine growth restriction (IUGR). PARTICIPANTS: Participants included women with uncomplicated pregnancies and pregnancies complicated by IUGR. RESULTS: Endothelial lipase (EL) and lipoprotein lipase (LPL) were the only lipases among the TLG family expressed in key cells of the human placenta. In first trimester, EL and LPL were expressed in trophoblasts. At term, EL was detected in trophoblasts and endothelial cells, whereas LPL was absent in these cells. Both lipases were found at placental blood vessels, EL in vascular endothelial cells and LPL in the surrounding smooth muscle cells. In total placental tissue EL expression prevails in first trimester and at term. Compared with normal placentas, EL mRNA was decreased (30%; P < 0.02), whereas LPL mRNA expression was increased (2.4-fold; P < 0.015) in IUGR. CONCLUSION: EL is the predominant TLG family member in the human placenta present at both interfaces. EL and LPL are dysregulated in IUGR.

Adhering maternal platelets can contribute to the cytokine and chemokine cocktail released by human first trimester villous placenta.

Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analyzing the inflammatory secretion profile of human placenta.

Reaction patterns of monoclonal antibodies to HLA-G in human tissues and on cell lines: a comparative study.

We compared the immunohistochemical reaction patterns of HLA-G-specific antibodies 87G, 4H84, G233, 16G1, and BFL.1 on human placentas under three different preparative conditions and on cryosections of other human tissues. Human and murine cell lines, either naturally expressing or transfected with HLA-G, were analyzed for their reaction patterns by immunocytochemistry and flow cytometry. Antibodies HCA2, TP25.99, W6/32 to classical HLA class I, anti-beta(2)-m and various non-HLA-G expressing cell lines were used as controls. The binding ability of the antibodies depends on the histotechnical procedure used. 4H84 and HCA2 bind to HLA-G despite aldehyde fixation and also paraffin embedding. 87G does not bind HLA-G in studies involving fixation with aldehydes. G233 labels HLA-G in aldehyde fixed but not paraffin embedded tissues. By immunocytochemistry HLA-G2 is merely detected with antibodies 4H84 and HCA2. MAb 16G1 binds to HLA-Gsol transfected cell lines only. The HLA-G specificity of mAb BFL.1 was considered as doubtful because it failed to react with most of the HLA-G transfected cell lines. Binding of 87G to the surface of monocytes or U-937 cells stimulated with IFN-gamma and GM-CSF is an Fc-receptor mediated phenomenon.

Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent. According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.

Immunohistochemical examination of trophoblast populations in human first trimester and term placentae and of first trimester spiral arteries with the monoclonal antibody GZ 112.

This paper presents an immunohistochemical study with a monoclonal mouse antibody GZ 112, an IgG1 kappa, which is directed against an antigen expressed in first trimester placenta by all proliferative and invasive extravillous trophoblast populations including a population of Langhans cells that represent extravillous stem cells. Additionally, the GZ 112 antigen is associated with morphological changes of spiral arteries preceding local trophoblast invasion. In term placentae, GZ 112 also strongly reacts with all extravillous trophoblast populations, but additionally recognizes partly villous cytotrophoblast and syncytiotrophoblast too, displaying a heterogeneous staining pattern. GZ 112 is directed against a 42-KDa antigen. Intracytoplasmic network-like staining and cross-reactivity with various human surface and glandular epithelia indicate a cytokeratin intermediate filament or a cytokeratin intermediate filament associated molecule as antigen.

The monoclonal antibody GZS-1 detects a maturation-associated antigen of human spermatozoa that is also present on the surface of human mononuclear blood cells.

A monoclonal antibody (GZS-1) has been generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunised with human sperm cells. The antibody was determined to be an IgG1. The corresponding antigen is present on the whole surface of ejaculated human spermatozoa. It is not detectable on spermatozoa of other mammalian species (rabbit, cat, dog, sheep, boar, bull, horse). In human male genital organs, immunostaining with GZS-1 is observed on sperm cells in the epididymis and the ductus deferens together with the lining epithelium of those organs. No reactivity of sperm cells or germ cell precursors in the testis has been detected. Functional tests using the antibody show a strong inhibitory effect of human sperm in the hamster egg penetration assay. Furthermore, the GZS-1 antigen is detectable on the surface of human lymphocytes and monocytes by immunogold electron microscopy and FACS analysis. By Western blotting of human sperm and seminal plasma performed under reducing conditions immunostaining was detected at 21-25, 31, 51-54, and 62 kDa. The reaction with human lymphocytes shows one major band at 62 kDa and additional bands at 31 and 54 kDa. The results suggest that the monoclonal antibody GZS-1 may recognise an antigen which is secreted from the epithelial cells of the epididymis and binds to ejaculated spermatozoa as a sperm coating antigen. This component may be involved in the capacitation of the sperm and the acrosome reaction. Molecules that are expressed both on sperm and on immunocompetent cells may be relevant for the regulation of immunological processes or for the development of the related immunological tolerance of sperm in the female reproductive tract.

The soluble pool of HLA-G produced by human trophoblasts does not include detectable levels of the intron 4-containing HLA-G5 and HLA-G6 isoforms.

In the context of implantation and pregnancy, several immunomodulating functions have been attributed to the different HLA-G isoforms. Increasing attention is now being addressed to the actively secreted soluble forms, because they might have a systemic function or could be useful as diagnostic tools. However, the cellular source of secretion, even during pregnancy, where HLA-G expression level is known to be highest, is still under debate. To elucidate the conflicting results, we investigated the isoform distribution in human first trimester and term placentas in situ and in vitro. Results obtained by applying immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR show that (1) all of the alpha1 domain-containing HLA-G isoforms are restrictedly expressed in the extravillous cytotrophoblasts (EVCTs) and very few first-trimester syncytiotrophoblasts, which directly cover cell columns, whereas mesenchymal cells of the villous chorion do not express HLA-G; (2) as demonstrated in western blots, trophoblasts express only the HLA-G1 isoform; (3) HLA-G5 and -G6 transcripts could be detected in human term placenta and isolated first-trimester trophoblasts but levels are extremely low; and (4) conditioned media of primary first-trimester trophoblasts, and the chorion laeve-derived trophoblastic cell line AC1-M59 do contain HLA-G1 fragments shed from the cell surface. Our data provide substantial evidence that none of the intron 4-containing isoforms, the so-called actively secreted, soluble HLA-G5 or -G6, are produced by human trophoblasts in situ or in vitro.

Paraffin-embedded manipulated blastocysts: a tool to demonstrate stem cell plasticity?

One of the big question marks in current stem cell research is whether there is true plasticity of adult progenitor cells (APC) or if cell fusion is the principle source of the supposed plasticity. The generation of chimeras by injecting adult progenitor cells into blastocysts is not new. This paper describes an efficient embedding technique for murine blastocysts injected with human APC. This method could help in establishing a novel tool to analyse the process of plasticity, if it truly exists. If this is the case, this technology could be of great help to characterize surface markers of stem cells in great detail. On the other hand, fusion of cells could also be investigated. A system of embedding blastocysts was set up using paraffin for further analysis by means of light microscopy and immunohistochemistry. The embedding of the chimaeras consists of fixing them first with paraformaldehyde in phosphate-buffered saline (PFA/PBS), embedding them in gelatine, fixing the gelatine block with PFA/PBS and finally fixing the gelatine block in a Petri dish by embedding it in paraffin. Using this protocol, the morphology of the blastocysts is well preserved.

The functionality of HLA-G is emerging.

In view of the recently published data, the HLA-G class Ib gene appears to be a functional locus. This is based on the following observations: 1) HLA-G is capable of presenting nonamer peptides and of exerting antigen-presenting functions; 2) HLA-G is a ligand for at least three natural killer (NK) and other cell inhibitory receptors of the immunoglobulin superfamily, namely leukocyte immunoglobulin-like receptor-1/immunoglobulin-like transcript (ILT)-2, ILT-4 and p49; 3) in addition to the extravillous cytotrophoblast cells, HLA-G proteins have been detected in endothelial cells of placental chorionic villi, as well as in amniotic fluid and in some medullary thymic epithelial cells; 4) major histocompatibility complex (MHC) class Ib genes that share the unique characteristics of HLA-G, including a high expression in placenta, have been reported in other mammalian species. In addition to the classical MHC class I roles (antigen presentation and ligation to NK receptors inducing inhibitory and/or activatory signals), HLA-G is likely to exert other, novel functions: first, HLA-G was shown to be involved in the control of HLA-E expression by furnishing the appropriate class I leader sequence nonamer peptide; second, we hypothesize that HLA-G could be a regulator of placental angiogenesis; third, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy. Such functional properties, although incompletely understood, are likely to be important in the outcome of human pregnancies but also in normal adult life.

HLA Class I protein expression in the human placenta.

The maternal tolerance to the semiallogeneic fetus is still a central theme in reproductive immunology. During placentation, fetally-derived, genetically dissimilar tissue and cells come into close contact with maternal tissue and cells, thus forming the so-called feto-maternal interface. The most extensive contact between fetally-derived and maternal blood cells is formed by the villous trophoblastic barrier, where the syncytiotrophoblast surface permanently floats in maternal blood. Further contact is made by some extravillous cytotrophoblast cells, either located at villous tips, in so-called cell islands, or the endovascular trophoblast population within the uteroplacental spiral arteries. The third contact zone is the so-called junctional zone within the decidua where the invading extravillous trophoblast cells encounter all maternal tissue leukocytes, which are mainly NK cells, macrophages and T cells; this junctional zone extends at the edge of the placenta to the amnio-chorionic membranes where the chorionic laeve trophoblast has intimate contact with decidua tissue. It is worth mentioning that evidence has shown that even in healthy pregnancies fetal and maternal lymphoid cells are able to transgress the trophoblastic barrier, which, anatomically, seems completely impermeable. Because of this intimate contact of foreign cells to the foreign immune system it is important to define the antigenic status of the placental cells, in particular with respect to antigens of the Major Histocompatibility complex. The role of the highly polymorphic classical class I molecules HLA -A, -B, -C, which are expressed on almost all somatic cells, is the induction of a specific immune response by presenting peptide antigens to T cells. In contrast, the non-classical HLA class I molecules HLA-G and HLA-E are thought to be involved in the induction of immune tolerance by acting as ligands for inhibitory receptors present on NK cells and macrophages. The non-classical HLA-E is also expressed ubiquitously, but HLA-G expression is characterized by a unique tissue expression mainly in the human placenta. A further feature of HLA-G is that its mRNA has undergone alternative splicing, resulting in at least 6 different isoforms, encoding different proteins: 4 membrane-bound and 2 soluble forms, which could simultaneously maintain different functions depending on their molecular structure. In our immunohistochemical study we investigated the expression of classical and non-classical HLA class I proteins in human placenta using various mAbs, which were kindly provided by the groups of A. Ziegler, D. Geraghty, O. Genbacev, MT. McMaster, A. King, YW. Loke and Ph. Le Bouteiller. For HLA-A,-B detection we used the antibody LA45; for detection of HLA-C,-B mAbs Tü149 and HC10. HLA-C expression alone was detected with mAb L31. HLA-G expression was studied using antibodies 4H84, G233, 87G, 16G1 and BFL.1. For HLA-E staining we used antibodies DT9 and V16. The classical HLA class I proteins are expressed in all non-trophoblastic cells including the fetal and maternal cells. Comparison of HLA-A and HLA-B staining intensities within the villous stroma indicates that during first trimester of pregnancy the fetal HLA-B proteins are expressed before HLA-A appears. Among the trophoblast populations, the syncytiotrophoblast does not show any HLA class I staining, but the extravillous cells express high amounts of HLA-G together with HLA-C. King and co-workers have shown recently, using methods other than immunohistochemistry, that first trimester extravillous trophoblast cells are also likely to express HLA-E. By contrast we did not detect HLA-E in any trophoblasts with antibodies DT9 and V16. There is still an ongoing and also controversial discussion about which kinds of cells in the placenta, other than extravillous trophoblast, express which kind of the HLA-G isoforms. Depending on the antibodies and the different immunohistochemical techniques used, different results have been described: Antibody 16G1 specific for soluble HLA-G labels syncytiotrophoblast, antibody BFL.1 endothelial cells of chorionic fetal blood vessels and antibody 87G Hofbauer cells. All these HLA-G labelings, apart from extravillous trophoblasts, are in complete contrast to the reaction pattern (merely extravillous trophoblast) given by antibody 4H84, which recognizes all HLA-G isoforms -including the soluble ones- through an epitope located on the a 1-domain of HLA-G. Future studies employing isoform-specific antibodies, which are not yet available for all of the possible isoforms, will elucidate the function and expression pattern of HLA-G in the human placenta.

Ultrastructural localization of insulin receptors in human placenta.

PROBLEM: Investigations to date on localization of placental insulin receptors (IR) at the ultrastructural level focused on the maternal facing side of the placenta but did not allow localization of IRs to intracellular compartments. METHOD: The ultrastructural localization of IR in placentae from early and term gestation was studied using the immunogold technique and a monoclonal antibody against the external domain of the IR (clone MA20). RESULTS: At 10 wk gestation, there were high levels of IR in both cytotrophoblast and syncytiotrophoblast, with sparse microvillous labeling. Fetal vessels contained various amounts of IR while stromal cells were also labeled. By 13 wk, this pattern was unchanged; however, at term, fewer IR were present in the syncytiotrophoblast, with more label on microvilli than before. Fetal vessels were well labeled, while stromal cells appeared unchanged. Intense labeling of fetal erythrocytes provided an internal positive control, while omission of the primary antiserum produced loss of binding. CONCLUSION: The data suggest a spatiotemporal alteration in placental IR distribution during pregnancy, possibly reflecting a shift in control of placental growth and metabolism from mother to fetus.

Immunohistochemical evidence of p53 protein in human placenta and choriocarcinoma cell lines.

We investigated the expression of the tumour-suppressor and cell cycle control protein p53 in human first trimester and term placenta, three choriocarcinoma cell lines (Jeg-3, JAR, BeWo) and human choriocarcinoma. Using monoclonal antibodies against p53 (DO-7, Ab-6, DO-1, PAb 1801), paraffin-embedded sections of first trimester and full-term placentae, human choriocarcinoma and Jeg-3, JAR and BeWo, as well as cytospins, were evaluated immunohistochemically. In addition, Western blots were carried out with the same antibodies on choriocarcinoma cell lines. In placentae, a small number of villous and extravillous cytotrophoblast cells, as well as very few syncytiotrophoblast cells, stained intensively. Also, p53 was visualized in some nuclei of the placental basal plate, whereas stroma and endothelium were negative for p53. Jeg-3, JAR and BeWo also showed a positive nuclear reaction with all applied antibodies. In paraffin-embedded sections of human choriocarcinoma, staining was confined to the nuclei of malignant cells. The results suggest that p53 is overexpressed not only in malignant tumour cells but in certain trophoblast cell populations of the human placenta as well.

First trimester human endovascular trophoblast cells express both HLA-C and HLA-G.

PROBLEM: In human pregnancies, trophoblasts, in contrast to placental connective tissue and the fetus itself, come into direct contact with the maternal allorecognizing system at special sites. Villous syncytiotrophoblasts washed around by maternal blood lack HLA class I proteins, whereas extravillous trophoblasts, which deeply invade maternal uterine tissues, express high amounts of HLA-G and also HLA-C, the latter to a lesser degree, however. A subpopulation of extravillous trophoblasts, the endovascular trophoblast, enters maternal spiral artery lumen and, like syncytiotrophoblast, comes into direct contact with maternal blood. Less is known about HLA class I distribution on this endovascular trophoblast subpopulation. METHOD OF STUDY: A comparative immununohistochemical analysis was done on decidual cryo-sections containing trophoblast-invaded spiral arteries using different anti-HLA class I monoclonal antibodies (mAbs) and a peroxidase-labeled streptavidinbiotin detection system. RESULTS: MAbs W6/32 (anti-HLA-A, -B, -C, -G), HCA2 (anti-HLA-A, -G) G233 and 87G (both anti-HLA-G) resulted in strong positivity on endovascular trophoblasts. L31 (anti-HLA-C) and HC10 (anti-HLA-B, -C) revealed clear positivity, whereas TU149 (anti-HLA-B, -C, some -A) produced a heterogeneous staining pattern, faintly positive on some endovascular trophoblastic cells and negative on others. MAb LA45 (anti-HLA-A, -B) did not bind to any endovascular trophoblast, neither did BFL.1 (anti-HLA-G) nor 16G1 (anti-HLA-G, soluble). CONCLUSION: This study shows that trophoblastic cells belonging to the endovascular subpopulation express considerable amounts of HLA-G and slightly less HLA-C.

Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages.

In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (-IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; -IP approximately 70%) as identified by rigorous immunocytochemistry. Most (approximately 70%) non-trophoblast cells in -IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-beta (hCG-beta) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-beta: 18-fold) in +IP than in -IP, with the exception of endothelin-1,2 (no change), angiotensin II (-70%) and 6-keto-prostaglandin-F1alpha (-40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in -IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.

Non-Michaelis-Menten kinetics of zero-trans glucose uptake by trophoblast cells from human term placentae and by choriocarcinoma (JEG-3/JAR) cells.

Maternal glucose is a major substrate for placental and fetal metabolism. The kinetics of its uptake into placental trophoblast cells has not been characterised yet and was therefore investigated in the present study. In addition to trophoblast cells isolated from human term placentae, JEG-3 and JAR choriocarcinoma cells were used. Measurements were carried out in 5 s intervals until 30 s with the non-metabolisable glucose analogue 3-O-[14C]methyl-D-glucose using confluent cells adhering to glass coverslips. L-[1-14C]glucose was used to correct for extracellular trapped tracer and diffusion. The uptake was rapid and saturable. It reached equilibrium after 30 s at 20 degrees C and could be inhibited by 0.4 mmol/l cytochalasin B up to 98%. The choriocarcinoma cells took up twice as much glucose as trophoblast cells. Fitting the experimental data to the Michaelis-Menten equation by non-linear regression failed to adequately describe the data, even when a contribution of diffusion to total uptake was considered. Introducing the Hill coefficient n into the Michaelis-Menten equation significantly improved the quality of the fits as was assessed by three statistical criteria. Using this equation modified for allosteric kinetics (v = k[To] [S]n)/(Km + [S]n)), parameters were calculated as Km = 12 mmol/l, Vmax = 17 fmol/l s-1 per cell, n = 1.1 for trophoblast cells; Km = 13 mmol/l, Vmax = 27 fmol/l s-1 per cell, n = 1.2 for JEG-3 cells and Km = 29 mmol/l, Vmax = fmol/l s-1 per cell, n = 1.4 for JAR cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Localisation of the high affinity facilitative glucose transporter protein GLUT 1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages.

In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum against a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reichert's membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.

Platelets contain interleukin-1 alpha and beta which are detectable on the cell surface after activation.

Activated platelets have been shown previously to exhibit membrane-bound IL-1 bioactivity, which leads to the question of localization of the cytokine in platelets. Using immunocytological and flow cytometric techniques, we found IL-1 alpha and IL-1 beta in the cytoplasma of both resting and thrombin-activated platelets. Immunogold-silver staining of the cell surface of activated platelets as well as preembedding antibody treatment of platelets revealed the presence of IL-1 (alpha and beta) in low density on the surface of intact cells in contrast to distinct enrichment in the cytoplasma of damaged platelets. Fibrin fibres present between cells indicated adsorbance of IL-1. There was also weak binding of anti-IL-1 alpha to the surface of thrombin-activated platelets as shown by flow cytometry. Following activation there appears to be some transfer of IL-1 onto the cell surface of activated cells, the bulk of the cytokine, however, is probably not released prior to platelet disintegration. In summary, we present evidence for the presence of both IL-1 alpha and IL-1 beta in resting and activated platelets without being able to demonstrate localization of the cytokines to specific subcellular structures.

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta.

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (Fc gamma RII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.